Huber R

References (17)

Title : Chemoproteomics-Enabled Identification of 4-Oxo-beta-Lactams as Inhibitors of Dipeptidyl Peptidases 8 and 9 - Carvalho_2022_Angew.Chem.Int.Ed.Engl_61_e202210498
Author(s) : Carvalho LAR , Ross B , Fehr L , Bolgi O , Wohrle S , Lum KM , Podlesainski D , Vieira AC , Kiefersauer R , Felix R , Rodrigues T , Lucas SD , Gross O , Geiss-Friedlander R , Cravatt BF , Huber R , Kaiser M , Moreira R
Ref : Angew Chem Int Ed Engl , : , 2022
Abstract : Dipeptidyl peptidases 8 and 9 (DPP8/9) have gathered interest as drug targets due to their important roles in biological processes like immunity and tumorigenesis. Elucidation of their distinct individual functions remains an ongoing task and could benefit from the availability of novel, chemically diverse and selective chemical tools. Here, we report the activity-based protein profiling (ABPP)-mediated discovery of 4-oxo-beta-lactams as potent, non-substrate-like nanomolar DPP8/9 inhibitors. X-ray crystallographic structures revealed different ligand binding modes for DPP8 and DPP9, including an unprecedented targeting of an extended S2' (eS2') subsite in DPP8. Biological assays confirmed inhibition at both target and cellular levels. Altogether, our integrated chemical proteomics and structure-guided small molecule design approach led to novel DPP8/9 inhibitors with alternative molecular inhibition mechanisms, delivering the highest selectivity index reported to date.
ESTHER : Carvalho_2022_Angew.Chem.Int.Ed.Engl_61_e202210498
PubMedSearch : Carvalho_2022_Angew.Chem.Int.Ed.Engl_61_e202210498
PubMedID: 36089535
Gene_locus related to this paper: human-DPP8 , human-DPP9

Title : Dipeptidyl peptidase 9 triggers BRCA2 degradation and promotes DNA damage repair - Bolgi_2022_EMBO.Rep__e54136
Author(s) : Bolgi O , Silva-Garcia M , Ross B , Pilla E , Kari V , Killisch M , Spitzner M , Stark N , Lenz C , Weiss K , Donzelli L , Gorrell MD , Grade M , Riemer J , Urlaub H , Dobbelstein M , Huber R , Geiss-Friedlander R
Ref : EMBO Rep , :e54136 , 2022
Abstract : N-terminal sequences are important sites for post-translational modifications that alter protein localization, activity, and stability. Dipeptidyl peptidase 9 (DPP9) is a serine aminopeptidase with the rare ability to cleave off N-terminal dipeptides with imino acid proline in the second position. Here, we identify the tumor-suppressor BRCA2 as a DPP9 substrate and show this interaction to be induced by DNA damage. We present crystallographic structures documenting intracrystalline enzymatic activity of DPP9, with the N-terminal Met1-Pro2 of a BRCA21-40 peptide captured in its active site. Intriguingly, DPP9-depleted cells are hypersensitive to genotoxic agents and are impaired in the repair of DNA double-strand breaks by homologous recombination. Mechanistically, DPP9 targets BRCA2 for degradation and promotes the formation of RAD51 foci, the downstream function of BRCA2. N-terminal truncation mutants of BRCA2 that mimic a DPP9 product phenocopy reduced BRCA2 stability and rescue RAD51 foci formation in DPP9-deficient cells. Taken together, we present DPP9 as a regulator of BRCA2 stability and propose that by fine-tuning the cellular concentrations of BRCA2, DPP9 alters the BRCA2 interactome, providing a possible explanation for DPP9's role in cancer.
ESTHER : Bolgi_2022_EMBO.Rep__e54136
PubMedSearch : Bolgi_2022_EMBO.Rep__e54136
PubMedID: 35912982
Gene_locus related to this paper: human-DPP8 , human-DPP9

Title : Aerosol-based ligand soaking of reservoir-free protein crystals - Ross_2021_J.Appl.Crystallogr_54_895
Author(s) : Ross B , Krapp S , Geiss-Friedlander R , Littmann W , Huber R , Kiefersauer R
Ref : J Appl Crystallogr , 54 :895 , 2021
Abstract : Soaking of macromolecular crystals allows the formation of complexes via diffusion of molecules into a preformed crystal for structural analysis. Soaking offers various advantages over co-crystallization, e.g. small samples and high-throughput experimentation. However, this method has disadvantages, such as inducing mechanical stress on crystals and reduced success rate caused by low affinity/solubility of the ligand. To bypass these issues, the Picodropper was previously developed in the authors' laboratory. This technique aimed to deliver small volumes of compound solution in response to crystal dehydration supported by the Free Mounting System humidity control or by IR-laser-induced protein crystal transformation. Herein, a new related soaking development, the Aerosol-Generator, is introduced. This device delivers compounds onto the solution-free surface of protein crystals using an ultrasonic technique. The produced aerosol stream enables an easier and more accurate control of solution volumes, reduced crystal handling, and crystal-size-independent soaking. The Aerosol-Generator has been used to produce complexes of DPP8 crystals, where otherwise regular soaking did not achieve complex formation. These results demonstrate the potential of this device in challenging ligand-binding scenarios and contribute to further understanding of DPP8 inhibitor binding.
ESTHER : Ross_2021_J.Appl.Crystallogr_54_895
PubMedSearch : Ross_2021_J.Appl.Crystallogr_54_895
PubMedID: 34188616
Gene_locus related to this paper: human-DPP8

Title : Dipeptidyl peptidase 9 triggers BRCA2 degradation by the N-degron pathway to promote DNA-damage repair - Silva-Garcia_2020_Biorxiv__
Author(s) : Silva-Garcia M , Bolgi O , Ross B , Pilla E , Kari V , Killisch M , Stark N , Lenz C , Spitzner M , Gorrell MD , Grade M , Urlaub H , Dobbelstein M , Huber R , Geiss-Friedlander R
Ref : Biorxiv , : , 2020
Abstract : Dipeptidyl peptidase 9 (DPP9) is a serine protease cleaving N-terminal dipeptides preferentially post-proline with (patho)physiological roles in the immune system and cancer. Only few DPP9 substrates are known. Here we identify an association of human DPP9 with the tumour suppressor BRCA2, a key player in repair of DNA double-strand breaks that promotes the formation of RAD51 filaments. This interaction is triggered by DNA-damage and requires access to the DPP9 active-site. We present crystallographic structures documenting the N-terminal Met1-Pro2 of a BRCA21-40 peptide captured in the DPP9 active-site. Mechanistically, DPP9 targets BRCA2 for degradation by the N-degron pathway, and promotes RAD51 foci formation. Both processes are phenocopied by BRCA2 N-terminal truncation mutants, indicating that DPP9 regulates both stability and the cellular stoichiometric interactome of BRCA2. Consistently, DPP9-deprived cells are hypersensitive to DNA-damage. Together, we identify DPP9 as a regulator of BRCA2, providing a possible explanation for DPP9 involvement in cancer development.
ESTHER : Silva-Garcia_2020_Biorxiv__
PubMedSearch : Silva-Garcia_2020_Biorxiv__
Gene_locus related to this paper: human-DPP8 , human-DPP9

Title : Structures and mechanism of dipeptidyl peptidases 8 and 9, important players in cellular homeostasis and cancer - Ross_2018_Proc.Natl.Acad.Sci.U.S.A_115_E1437
Author(s) : Ross B , Krapp S , Augustin M , Kierfersauer R , Arciniega M , Geiss-Friedlander R , Huber R
Ref : Proc Natl Acad Sci U S A , 115 :E1437 , 2018
Abstract : Dipeptidyl peptidases 8 and 9 are intracellular N-terminal dipeptidyl peptidases (preferentially postproline) associated with pathophysiological roles in immune response and cancer biology. While the DPP family member DPP4 is extensively characterized in molecular terms as a validated therapeutic target of type II diabetes, experimental 3D structures and ligand-/substrate-binding modes of DPP8 and DPP9 have not been reported. In this study we describe crystal and molecular structures of human DPP8 (2.5 A) and DPP9 (3.0 A) unliganded and complexed with a noncanonical substrate and a small molecule inhibitor, respectively. Similar to DPP4, DPP8 and DPP9 molecules consist of one beta-propeller and alpha/beta hydrolase domain, forming a functional homodimer. However, they differ extensively in the ligand binding site structure. In intriguing contrast to DPP4, where liganded and unliganded forms are closely similar, ligand binding to DPP8/9 induces an extensive rearrangement at the active site through a disorder-order transition of a 26-residue loop segment, which partially folds into an alpha-helix (R-helix), including R160/133, a key residue for substrate binding. As vestiges of this helix are also seen in one of the copies of the unliganded form, conformational selection may contributes to ligand binding. Molecular dynamics simulations support increased flexibility of the R-helix in the unliganded state. Consistently, enzyme kinetics assays reveal a cooperative allosteric mechanism. DPP8 and DPP9 are closely similar and display few opportunities for targeted ligand design. However, extensive differences from DPP4 provide multiple cues for specific inhibitor design and development of the DPP family members as therapeutic targets or antitargets.
ESTHER : Ross_2018_Proc.Natl.Acad.Sci.U.S.A_115_E1437
PubMedSearch : Ross_2018_Proc.Natl.Acad.Sci.U.S.A_115_E1437
PubMedID: 29382749
Gene_locus related to this paper: human-DPP8 , human-DPP9

Title : Rigidity and flexibility of dipeptidyl peptidase IV: crystal structures of and docking experiments with DPIV - Engel_2006_J.Mol.Biol_355_768
Author(s) : Engel M , Hoffmann T , Manhart S , Heiser U , Chambre S , Huber R , Demuth HU , Bode W
Ref : Journal of Molecular Biology , 355 :768 , 2006
Abstract : Dipeptidyl peptidase IV (DPIV) is an alpha,beta-hydrolase-like serine exopeptidase, which removes dipeptides, preferentially with a C-terminal l-Pro residue, from the N terminus of longer peptide substrates. Previously, we determined the tetrameric 1.8A crystal structure of native porcine DPIV. Each monomer is composed of a beta-propeller and a catalytic domain, which together embrace an internal cavity housing the active centre. This cavity is connected to the bulk solvent by a "propeller opening" and a "side opening". Here, we analyse DPIV complexes with a t-butyl-Gly-Pro-Ile tripeptide, Pro-boroPro, a piperazine purine compound, and aminoethyl phenyl sulfonylfluoride. The latter two compounds bind to the active-site groove in a compact and a quite bulky manner, respectively, causing considerable shifts of the catalytic Ser630 side-chain and of the Tyr547 phenolic group, which forms the oxyanion hole. The tripeptide, mimicking a peptide substrate, is clamped to the active site through tight interactions via its N-terminal alpha-ammonium group, the P2 carbonyl group, the P1-l-Pro side-chain, the C-terminal carboxylate group, and the stable orthoacid ester amide formed between the scissile peptide carbonyl group and Ser630 O(gamma). This stable trapping of the tripeptide could be due to stabilization of the protonated His740 imidazolium cation by the adjacent negatively charged C-terminal carboxylate group, preventing proton transfer to the leaving group nitrogen atom. Docking experiments with the compact rigid 58 residue protein aprotinin, which had been shown to be processed by DPIV, indicate that the Arg1-Pro2 N terminus can access the DPIV active site only upon widening of its side openings, probably by separation of the first and the last propeller blades, and/or of the catalytic and the propeller domain.
ESTHER : Engel_2006_J.Mol.Biol_355_768
PubMedSearch : Engel_2006_J.Mol.Biol_355_768
PubMedID: 16330047
Gene_locus related to this paper: pig-dpp4

Title : X-ray snapshots of peptide processing in mutants of tricorn-interacting factor F1 from Thermoplasma acidophilum - Goettig_2005_J.Biol.Chem_280_33387
Author(s) : Goettig P , Brandstetter H , Groll M , Gohring W , Konarev PV , Svergun DI , Huber R , Kim JS
Ref : Journal of Biological Chemistry , 280 :33387 , 2005
Abstract : The tricorn-interacting factor F1 of the archaeon Thermoplasma acidophilum cleaves small hydrophobic peptide products of the proteasome and tricorn protease. F1 mutants of the active site residues that are involved in substrate recognition and catalysis displayed distinct activity patterns toward fluorogenic test substrates. Crystal structures of the mutant proteins complexed with peptides Phe-Leu, Pro-Pro, or Pro-Leu-Gly-Gly showed interaction of glutamates 213 and 245 with the N termini of the peptides and defined the S1 and S1' sites and the role of the catalytic residues. Evidence was found for processive peptide cleavage in the N-to-C direction, whereby the P1' product is translocated into the S1 site. A functional interaction of F1 with the tricorn protease was observed with the inactive F1 mutant G37A. Moreover, small angle x-ray scattering measurements for tricorn and inhibited F1 have been interpreted as formation of transient and substrate-induced complexes.
ESTHER : Goettig_2005_J.Biol.Chem_280_33387
PubMedSearch : Goettig_2005_J.Biol.Chem_280_33387
PubMedID: 15994304
Gene_locus related to this paper: theac-pip

Title : The genome sequence of the extreme thermophile Thermus thermophilus - Henne_2004_Nat.Biotechnol_22_547
Author(s) : Henne A , Bruggemann H , Raasch C , Wiezer A , Hartsch T , Liesegang H , Johann A , Lienard T , Gohl O , Martinez-Arias R , Jacobi C , Starkuviene V , Schlenczeck S , Dencker S , Huber R , Klenk HP , Kramer W , Merkl R , Gottschalk G , Fritz HJ
Ref : Nat Biotechnol , 22 :547 , 2004
Abstract : Thermus thermophilus HB27 is an extremely thermophilic, halotolerant bacterium, which was originally isolated from a natural thermal environment in Japan. This organism has considerable biotechnological potential; many thermostable proteins isolated from members of the genus Thermus are indispensable in research and in industrial applications. We present here the complete genome sequence of T. thermophilus HB27, the first for the genus Thermus. The genome consists of a 1,894,877 base pair chromosome and a 232,605 base pair megaplasmid, designated pTT27. The 2,218 identified putative genes were compared to those of the closest relative sequenced so far, the mesophilic bacterium Deinococcus radiodurans. Both organisms share a similar set of proteins, although their genomes lack extensive synteny. Many new genes of potential interest for biotechnological applications were found in T. thermophilus HB27. Candidates include various proteases and key enzymes of other fundamental biological processes such as DNA replication, DNA repair and RNA maturation.
ESTHER : Henne_2004_Nat.Biotechnol_22_547
PubMedSearch : Henne_2004_Nat.Biotechnol_22_547
PubMedID: 15064768
Gene_locus related to this paper: thet2-q72hm9 , thet2-q72hv6 , thet2-q72hz1 , thet2-q72i91 , thet2-q72j75 , thet2-q72jk9 , thet2-q72jm3 , thet2-q72kf4 , thet2-q72kp8 , thet2-q746k7 , theth-metx , theth-TT1662 , theth-TTC1787

Title : The crystal structure of dipeptidyl peptidase IV (CD26) reveals its functional regulation and enzymatic mechanism - Engel_2003_Proc.Natl.Acad.Sci.U.S.A_100_5063
Author(s) : Engel M , Hoffmann T , Wagner L , Wermann M , Heiser U , Kiefersauer R , Huber R , Bode W , Demuth HU , Brandstetter H
Ref : Proc Natl Acad Sci U S A , 100 :5063 , 2003
Abstract : The membrane-bound glycoprotein dipeptidyl peptidase IV (DP IV, CD26) is a unique multifunctional protein, acting as receptor, binding and proteolytic molecule. We have determined the sequence and 1.8 A crystal structure of native DP IV prepared from porcine kidney. The crystal structure reveals a 2-2-2 symmetric tetrameric assembly which depends on the natively glycosylated beta-propeller blade IV. The crystal structure indicates that tetramerization of DP IV is a key mechanism to regulate its interaction with other components. Each subunit comprises two structural domains, the N-terminal eight-bladed beta-propeller with open Velcro topology and the C-terminal alpha/beta-hydrolase domain. Analogy with the structurally related POP and tricorn protease suggests that substrates access the buried active site through the beta-propeller tunnel while products leave the active site through a separate side exit. A dipeptide mimicking inhibitor complexed to the active site discloses key determinants for substrate recognition, including a Glu-Glu motif that distinguishes DP IV as an aminopeptidase and an oxyanion trap that binds and activates the P(2)-carbonyl oxygen necessary for efficient postproline cleavage. We discuss active and nonactive site-directed inhibition strategies of this pharmaceutical target protein.
ESTHER : Engel_2003_Proc.Natl.Acad.Sci.U.S.A_100_5063
PubMedSearch : Engel_2003_Proc.Natl.Acad.Sci.U.S.A_100_5063
PubMedID: 12690074
Gene_locus related to this paper: pig-dpp4

Title : Structures of the tricorn-interacting aminopeptidase F1 with different ligands explain its catalytic mechanism - Goettig_2002_Embo.J_21_5343
Author(s) : Goettig P , Groll M , Kim JS , Huber R , Brandstetter H
Ref : EMBO Journal , 21 :5343 , 2002
Abstract : F1 is a 33.5 kDa serine peptidase of the alpha/beta-hydrolase family from the archaeon Thermoplasma acidophilum. Subsequent to proteasomal protein degradation, tricorn generates small peptides, which are cleaved by F1 to yield single amino acids. We have solved the crystal structure of F1 with multiwavelength anomalous dispersion (MAD) phasing at 1.8 A resolution. In addition to the conserved catalytic domain, the structure reveals a chiefly alpha-helical domain capping the catalytic triad. Thus, the active site is accessible only through a narrow opening from the protein surface. Two structures with molecules bound to the active serine, including the inhibitor phenylalanyl chloromethylketone, elucidate the N-terminal recognition of substrates and the catalytic activation switch mechanism of F1. The cap domain mainly confers the specificity for hydrophobic side chains by a novel cavity system, which, analogously to the tricorn protease, guides substrates to the buried active site and products away from it. Finally, the structure of F1 suggests a possible functional complex with tricorn that allows efficient processive degradation to free amino acids for cellular recycling.
ESTHER : Goettig_2002_Embo.J_21_5343
PubMedSearch : Goettig_2002_Embo.J_21_5343
PubMedID: 12374735
Gene_locus related to this paper: theac-pip

Title : Crystal structure of the tricorn protease reveals a protein disassembly line - Brandstetter_2001_Nature_414_466
Author(s) : Brandstetter H , Kim JS , Groll M , Huber R
Ref : Nature , 414 :466 , 2001
Abstract : The degradation of cytosolic proteins is carried out predominantly by the proteasome, which generates peptides of 7-9 amino acids long. These products need further processing. Recently, a proteolytic system was identified in the model organism Thermoplasma acidophilum that performs this processing. The hexameric core protein of this modular system, referred to as tricorn protease, is a 720K protease that is able to assemble further into a giant icosahedral capsid, as determined by electron microscopy. Here, we present the crystal structure of the tricorn protease at 2.0 A resolution. The structure reveals a complex mosaic protein whereby five domains combine to form one of six subunits, which further assemble to form the 3-2-symmetric core protein. The structure shows how the individual domains coordinate the specific steps of substrate processing, including channelling of the substrate to, and the product from, the catalytic site. Moreover, the structure shows how accessory protein components might contribute to an even more complex protein machinery that efficiently collects the tricorn-released products.
ESTHER : Brandstetter_2001_Nature_414_466
PubMedSearch : Brandstetter_2001_Nature_414_466
PubMedID: 11719810

Title : The complete genome of the hyperthermophilic bacterium Aquifex aeolicus - Deckert_1998_Nature_392_353
Author(s) : Deckert G , Warren PV , Gaasterland T , Young WG , Lenox AL , Graham DE , Overbeek R , Snead MA , Keller M , Aujay M , Huber R , Feldman RA , Short JM , Olsen GJ , Swanson RV
Ref : Nature , 392 :353 , 1998
Abstract : Aquifex aeolicus was one of the earliest diverging, and is one of the most thermophilic, bacteria known. It can grow on hydrogen, oxygen, carbon dioxide, and mineral salts. The complex metabolic machinery needed for A. aeolicus to function as a chemolithoautotroph (an organism which uses an inorganic carbon source for biosynthesis and an inorganic chemical energy source) is encoded within a genome that is only one-third the size of the E. coli genome. Metabolic flexibility seems to be reduced as a result of the limited genome size. The use of oxygen (albeit at very low concentrations) as an electron acceptor is allowed by the presence of a complex respiratory apparatus. Although this organism grows at 95 degrees C, the extreme thermal limit of the Bacteria, only a few specific indications of thermophily are apparent from the genome. Here we describe the complete genome sequence of 1,551,335 base pairs of this evolutionarily and physiologically interesting organism.
ESTHER : Deckert_1998_Nature_392_353
PubMedSearch : Deckert_1998_Nature_392_353
PubMedID: 9537320
Gene_locus related to this paper: aquae-AQ.1571 , aquae-aq327 , aquae-aq2138 , aquae-dlhh

Title : The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C - Gomis-Ruth_1995_Embo.J_14_4387
Author(s) : Gomis-Ruth FX , Gomez M , Bode W , Huber R , Aviles FX
Ref : EMBO Journal , 14 :4387 , 1995
Abstract : The metalloexozymogen procarboxypeptidase A is mainly secreted in ruminants as a ternary complex with zymogens of two serine endoproteinases, chymotrypsinogen C and proproteinase E. The bovine complex has been crystallized, and its molecular structure analysed and refined at 2.6 A resolution to an R factor of 0.198. In this heterotrimer, the activation segment of procarboxypeptidase A essentially clamps the other two subunits, which shield the activation sites of the former from tryptic attack. In contrast, the propeptides of both serine proproteinases are freely accessible to trypsin. This arrangement explains the sequential and delayed activation of the constituent zymogens. Procarboxypeptidase A is virtually identical to the homologous monomeric porcine form. Chymotrypsinogen C displays structural features characteristic for chymotrypsins as well as elastases, except for its activation domain; similar to bovine chymotrypsinogen A, its binding site is not properly formed, while its surface located activation segment is disordered. The proproteinase E structure is fully ordered and strikingly similar to active porcine elastase; its specificity pocket is occluded, while the activation segment is fixed to the molecular surface. This first structure of a native zymogen from the proteinase E/elastase family does not fundamentally differ from the serine proproteinases known so far.
ESTHER : Gomis-Ruth_1995_Embo.J_14_4387
PubMedSearch : Gomis-Ruth_1995_Embo.J_14_4387
PubMedID: 7556081

Title : Lipoprotein lipase deficiency due to a 3' splice site mutation in intron 6 of the lipoprotein lipase gene - Holzl_1994_J.Lipid.Res_35_2161
Author(s) : Holzl B , Huber R , Paulweber B , Patsch JR , Sandhofer F
Ref : J Lipid Res , 35 :2161 , 1994
Abstract : In a patient with primary hyperchylomicronemia as a result of lipoprotein lipase (LPL) deficiency, we sequenced all translated exons and intron-exon boundaries of the LPL gene. We found a C-->A mutation in position -3 at the acceptor splice site of intron 6 which caused aberrant splicing. The major transcript showed a deletion of exons 6 through 9 and amounted to about 3% of the normal transcript of a healthy control individual. In addition to this major transcript, we found trace amounts of both a normally spliced LPL mRNA and a second aberrant transcript devoid of exon 7. On the same allele, we detected in the LPL gene of our patient four polymorphic variations, three of which have not as yet been described. A second patient from an unrelated family, but from the same geographic area, was also found to be homozygous for the same mutation. Of the relatives of the two probands studied, 11 were heterozygous and 5 were unaffected by the mutation. LPL activity in postheparin plasma was near zero in the probands and reduced in 4 of the 10 heterozygotes. A third hyperchylomicronemic patient from the same area was found to be a compound heterozygote who carried on one allele the 3' splice site mutation of intron 6 and on the other one an already described missense mutation resulting in Gly188-->Glu substitution.
ESTHER : Holzl_1994_J.Lipid.Res_35_2161
PubMedSearch : Holzl_1994_J.Lipid.Res_35_2161
PubMedID: 7897314

Title : Three-dimensional structure of porcine pancreatic procarboxypeptidase A. A comparison of the A and B zymogens and their determinants for inhibition and activation - Guasch_1992_J.Mol.Biol_224_141
Author(s) : Guasch A , Coll M , Aviles FX , Huber R
Ref : Journal of Molecular Biology , 224 :141 , 1992
Abstract : The three-dimensional structure of procarboxypeptidase A (PCPA) from porcine pancreas has been determined at 2 A resolution and refined to a crystallographic R-factor of 0.198, with a root-mean-square deviation from ideal values for bond lengths of 0.015 A and for angles of 2.1 degrees. It is compared with procarboxypeptidase B (PCPB) from the same tissue. The 94/95 residue activation segments of PCPA/PCPB have equivalent folds: an N-terminal globular region with an open sandwich antiparallel alpha/antiparallel beta topology, followed by an extended alpha-helical segment, the connection to the enzyme. Alignment of the secondary structures of the activation segments of PCPA and PCPB (residues A1 to A99) indicates a two residue insertion between residues A34 and A35 and a C-terminal helix that is two turns longer in PCPA compared to PCPB. A deletion is observed between residues A43 to A45, the region containing the short 3(10) helix that covers the active site in PCPB. The globular region (A4 to A80) shields the preformed active center of carboxypeptidase A (CPA), but none of the residues involved in catalysis makes direct contacts with the activation segment. In contrast, subsites S2, S3 and S4 of the enzyme, involved in the binding of peptidic substrates, are blocked by specific contacts with residues AspA36, TrpA38, ArgA47, AspA53 and GluA86 of the activation segment. It has been described that several residues of CPA exhibit different conformations in the free enzyme compared to when substrate is bound: Arg127, Arg145, Glu270 and Tyr248. In PCPA all of these residues are found in the "active" conformation, as if substrate were actually bound. The presence of a ligand, tentatively interpreted as a free amino acid (Val) in the active center could explain this fact. The connecting region (A80 to A99), the target for proteolytic activation, establishes fewer contacts with the enzyme in PCPA than in PCPB. The activation segment of PCPA (A4 to A99) remains bound to the enzyme after the first trypsin cleavage between ArgA99-Ala1 probably due to the stability conferred on it by the alpha-helix (alpha 3) of the connecting segment. These and other structural features may explain the differences in intrinsic activity and different rates or proteolytic activation of each zymogen.
ESTHER : Guasch_1992_J.Mol.Biol_224_141
PubMedSearch : Guasch_1992_J.Mol.Biol_224_141
PubMedID: 1548696

Title : Three-dimensional structure of porcine procarboxypeptidase B: a structural basis of its inactivity - Coll_1991_Embo.J_10_1
Author(s) : Coll M , Guasch A , Aviles FX , Huber R
Ref : EMBO Journal , 10 :1 , 1991
Abstract : Procarboxypeptidase B is converted to enzymatically active carboxypeptidase B by limited proteolysis catalysed by trypsin, removing the long N-terminal activation segment of 95 amino acids. The three-dimensional crystal structure of procarboxypeptidase B from porcine pancreas has been determined at 2.3 A resolution and refined to a crystallographic R-factor of 0.169. The functional determinants of its enzymatic inactivity and of its activation by limited proteolysis have thus been unveiled. The activation segment folds in a globular region with an open sandwich antiparallel-alpha antiparallel-beta topology and in a C terminal alpha-helix which connects it to the enzyme moiety. The globular region (A7-A82) shields the preformed active site, and establishes specific interactions with residues important for substrate recognition. AspA41 forms a salt bridge with Arg145, which in active carboxypeptidase binds the C-terminal carboxyl group of substrate molecules. The connecting region occupies the putative extended substrate binding site. The scissile peptide bond cleaved by trypsin during activation is very exposed. Its cleavage leads to the release of the activation segment and to exposure of the substrate binding site. An open-sandwich folding has been observed in a number of other proteins and protein domains. One of them is the C-terminal fragment of L7/L12, a ribosomal protein from Escherichia coli that displays a topology similar to the activation domain of procarboxypeptidase.
ESTHER : Coll_1991_Embo.J_10_1
PubMedSearch : Coll_1991_Embo.J_10_1
PubMedID: 1989878

Title : Tracing a 14C-labeled carbamate molluscicide through the digestive system of deroceras reticulatum (muller) - Triebskorn_1990_Pest.Sci_28_321
Author(s) : Triebskorn R , Knast C , Huber R , Brem G
Ref : Pest Sci , 28 :321 , 1990
Abstract : The passage of a 14C-labeled carbamate, 2-(2-chloro-1-methoxy-' ethoxy)phenyl N-methylcarbamate, or its labeled metabolites through the alimentary system of the grey garden slug Deroceras reticulatum was examined by autoradiographic studies and scintillation counting. It was demonstrated that, in a first step, the molluscicide penetrated the cells of the oesophagus and the crop. It was quickly transported by the hemolymph to the periphery of the body and re-entered the cells of the digestive tract and the mid-gut gland in a second step from the hemolymph side. The crypt cells of the mid-gut gland are discussed as cells involved in detoxification, and connective tissue cells as the major storage sites of the labeled material. Excretion in feces and secretion in mucus are thought to be the routes of 14C elimination.
ESTHER : Triebskorn_1990_Pest.Sci_28_321
PubMedSearch : Triebskorn_1990_Pest.Sci_28_321